There have been reported several derivatization regents for use in determination of .alpha.-keto acids by spectroscopic or fluorescent methods in connection with liquid chromatography.
C. Hemming and C. J. Gubler proposed to determine an .alpha.-keto acid by a spectroscopic method using 2,4-dinitrophenylhydrazine (Anal. Biochem. 92:31, 1979). Regarding the determination of keto acids by a fluorescent method, on the other hand, some workers have reported using 4'-hydrazino-2-stilbazole (cf. T. Hirata, M. Kai, K. Kohashi and Y. Ohkura, J. Chromatogr. 226:25, 1981) and o-phenylenediamine (cf. J. C. Liao, N. E. Hoffman, J. J. Barboriak and D. A. Roth, Clin. Chem. 2:802, 1977; and T. Hayashi, H,. Tsuchiya and H. Naruse, J. Chromatogr. 273:245, 1983). Although each of these reported methods fives a substantially high sensitivity, it is impossible thereby to determine an .alpha.-keto acid at a femtomol (fmcl: 10.sup.-15 mol) level.
The present inventors formerly synthesized 1,2-diamino-4,5-dimethoxybenzene and 1,2-diamino-4,5-methylenedioxybenzene each as a fluorescent analysis reagent for .alpha.-keto acids having a high sensitivity and a high selectivity. For the former reagent, refer to S. Hara, Y. Takemori, T. Iwata, M. Yamaguchi, M. Nakamura and Y. Ohkura, Anal. Chem. Acta. 172:167, 1985; and S. Hara, M. Yaaguchi, M. Nakamura and Y. Ohkura, Chem. Pharm. Bull. 33:3493, 1985. For the latter reagent, reference is made to M. Nakamura, S. Hara, M. Yamaguchi, Y. Takemori and Y. Ohkura, Chem. Pharm. Bull. 35:687, 1985. The present inventors have attempted to apply these reagents to liquid chromatography and have thus succeeded in determining .alpha.-keto acids at a femtomol level. These reagents have been used for determining .alpha.-keto acids contained in human urine and serum, as disclosed in S. Hara, Y. Takemori, M. Yamaguchi, N. Nakamura and Y. Ohkura, J. Chromatog. 344:33, 1985.
Recently, techniques for chemiluminescent detection have been introduced into liquid chromatographic analyses. As a result, there have been reported several reagents which react with amino acids, amine or carboxylic acids and thus convert these compounds into chemiluminescent derivatives. For reagents for amino acids, refer to S. R. Spurlin and M. M. Cooper, Anal. Lett. 19:2277, 1986; reagents for amines, T. Kawasaki, M. Maeda and A. Tsuji, J. Chromatogr. 328:121, 1985. For reagents for carboxylic acids, refer to T. Kawasaki, M. Maeda, and A. Tsuji, J. Chromatogr. 328:121, 1985; H. Yuki, H. Azuma, M. Maeda and H Kawasaki, Chem. Pharm. Bull. 36:1905, 1988; and H. Karatani, J. Takano, S. Morishita, M. Yoshida and M. Sato, Bunseki Kagaku 38:59, 1989.
As described above, attempts have been recently made to determine .alpha.-keto acids by spectroscopic or fluorescent methods. However, there has been reported hitherto no reagent required for sensitively and selectively determining a trace amount of an .alpha.-keto acid by using chemiluminescence.
The present inventor has conducted extensive studies on a reagent whereby a trace amount of an .alpha.-keto acid can be selectively determined by taking advantage of chemiluminescence. As a result, he has discovered that intense chemiluminescence is generated by reacting a quinoxalinone compound, which is obtained by the reaction of an keto acid with 4,5-diaminophthalhydrazide dihydrochloride (hereinafter referred to simply as 4,5-DPJ:2HCl), with hydrogen peroxide in an alkaline polar medium in the presence of potassium hexacyanoferrate (III). Thus, he has separately applied for a patent based upon this finding, on the same date as the parent of the present application, as a novel process for determining an keto acid.
The above chemical reaction may be represented by the following formula: ##STR9## Note: *:R represents a group selected from the group consisting of hydrogen, amino, carboxyl, thiol, n-propyl, isopropyl, n-butyl, sec-butyl, benzyl, carboxyethyl, tert-butyl, and
wherein
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